Multiplexed peptide analysis for kinetic measurements of major human apolipoproteins by LC/MS/MS
2016; Elsevier BV; Volume: 57; Issue: 3 Linguagem: Inglês
10.1194/jlr.d064618
ISSN1539-7262
AutoresMikaël Croyal, F. Fall, Véronique Ferchaud‐Roucher, Maud Chétiveaux, Yassine Zaïr, Khadija Ouguerram, Michel Krempf, Estelle Nobécourt,
Tópico(s)Mass Spectrometry Techniques and Applications
ResumoA multiplexed assay was developed by MS to analyze, in a single run, six major human Apos involved in lipoprotein metabolism: ApoA-I, ApoA-II, ApoB100, ApoC-II, ApoC-III, and ApoE. This method was validated in vivo in six subjects who received a 14 h constant infusion of [5,5,5-2H3]L-leucine at 10 μM/kg/h. Plasma lipoprotein fractions were isolated from collected blood samples and were digested with trypsin. Proteotypic peptides were subsequently analyzed by LC/MS/MS. Enrichment measurement data were compared with those obtained by the standard method using GC/MS. The required time to obtain the LC/MS/MS data was less than that needed for GC/MS. The enrichments from both methods were correlated for ApoA-I (r = 0.994; P < 0.0001) and ApoB100 (r = 0.999; P < 0.0001), and the Bland-Altman plot confirmed the similarity of the two methods. Intra- and inter-assay variability calculated for the six Apos of interest did not exceed 10.7 and 12.5%, respectively, and kinetic parameters were similar and/or in agreement with previously reported data. Therefore, LC/MS/MS can be considered as a useful tool for human Apo kinetic studies using stable isotopes. A multiplexed assay was developed by MS to analyze, in a single run, six major human Apos involved in lipoprotein metabolism: ApoA-I, ApoA-II, ApoB100, ApoC-II, ApoC-III, and ApoE. This method was validated in vivo in six subjects who received a 14 h constant infusion of [5,5,5-2H3]L-leucine at 10 μM/kg/h. Plasma lipoprotein fractions were isolated from collected blood samples and were digested with trypsin. Proteotypic peptides were subsequently analyzed by LC/MS/MS. Enrichment measurement data were compared with those obtained by the standard method using GC/MS. The required time to obtain the LC/MS/MS data was less than that needed for GC/MS. The enrichments from both methods were correlated for ApoA-I (r = 0.994; P < 0.0001) and ApoB100 (r = 0.999; P < 0.0001), and the Bland-Altman plot confirmed the similarity of the two methods. Intra- and inter-assay variability calculated for the six Apos of interest did not exceed 10.7 and 12.5%, respectively, and kinetic parameters were similar and/or in agreement with previously reported data. Therefore, LC/MS/MS can be considered as a useful tool for human Apo kinetic studies using stable isotopes. Lipoprotein kinetic studies using radioactive or stable isotope tracers have been performed for years in humans to gain a better understanding of the mechanisms involved in lipid metabolism disturbances and related diseases (1Foster D.M. Barrett P.H. Toffolo G. Beltz W.F. Cobelli C. Estimating the fractional synthetic rate of plasma apolipoproteins and lipids from stable isotope data.J. Lipid Res. 1993; 34: 2193-2205Abstract Full Text PDF PubMed Google Scholar, 2Lam S.M. Shui G. Lipidomics as a principal tool for advancing biomedical research.J. Genet. Genomics. 2013; 40: 375-390Crossref PubMed Scopus (86) Google Scholar). Endogenous labeling with an amino acid tracer of Apo, a main component of lipoproteins, is commonly used, assuming the kinetics of Apos represent a good estimate of those of the entire lipoprotein metabolism (1Foster D.M. Barrett P.H. Toffolo G. Beltz W.F. Cobelli C. Estimating the fractional synthetic rate of plasma apolipoproteins and lipids from stable isotope data.J. Lipid Res. 1993; 34: 2193-2205Abstract Full Text PDF PubMed Google Scholar, 3Lichtenstein A.H. Cohn J.S. Hachey D.L. Millar J.S. Ordovas J.M. Schaefer E.J. Comparison of deuterated leucine, valine, and lysine in the measurement of human apolipoprotein A-I and B-100 kinetics.J. Lipid Res. 1990; 31: 1693-1701Abstract Full Text PDF PubMed Google Scholar). The incorporation of a labeled amino acid into the protein during its synthesis is subsequently measured over time and analyzed with a compartmental model to obtain the kinetic data (4Barrett P.H. Watts G.F. Kinetic studies of lipoprotein metabolism in the metabolic syndrome including effects of nutritional interventions.Curr. Opin. Lipidol. 2003; 14: 61-68Crossref PubMed Scopus (30) Google Scholar, 5Matthan N.R. Jalbert S.M. Lamon-Fava S. Dolnikowski G.G. Welty F.K. Barrett H.R. Schaefer E.J. Lichtenstein A.H. TRL, IDL, and LDL apolipoprotein B-100 and HDL apolipoprotein A-I kinetics as a function of age and menopausal status.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1691-1696Crossref PubMed Scopus (27) Google Scholar). The reference method to measure tracer enrichments involves isolation of the Apos by gel electrophoresis followed by acid hydrolysis to obtain unlabeled and labeled amino acids. Then, the amino acids are derivatized for GC/MS analysis (3Lichtenstein A.H. Cohn J.S. Hachey D.L. Millar J.S. Ordovas J.M. Schaefer E.J. Comparison of deuterated leucine, valine, and lysine in the measurement of human apolipoprotein A-I and B-100 kinetics.J. Lipid Res. 1990; 31: 1693-1701Abstract Full Text PDF PubMed Google Scholar). These approaches are limited to one or a small number of relatively abundant Apos and remain a time-consuming process. Kinetic studies are therefore mainly focused on the most abundant structural Apos (ApoA-I and ApoB100) and less on others, although they have a central role in lipid metabolism (ApoC-II, ApoC-III and ApoE) (6Pan Y. Zhou H. Mahsut A. Rohm R.J. Berejnaia O. Price O. Chen Y. Castro-Perez J. Lassman M.E. McLaren D. et al.Static and turnover kinetic measurement of protein biomarkers involved in triglyceride metabolism including apoB48 and apoA5 by LC/MS/MS.J. Lipid Res. 2014; 55: 1179-1187Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The combination of proteomic tools, such as enzymatic proteolysis and liquid LC/MS/MS, has appeared recently to be a powerful tool to study plasma proteins (7Lee A.Y. Yates N.A. Ichetovkin M. Deyanova E. Southwick K. Fisher T.S. Wang W. Loderstedt J. Walker N. Zhou H. et al.Measurement of fractional synthetic rates of multiple protein analytes by triple quadrupole mass spectrometry.Clin. Chem. 2012; 58: 619-627Crossref PubMed Scopus (25) Google Scholar, 8Lassman M.E. McLaughlin T.M. Somers E.P. Stefanni A.C. Chen Z. Murphy B.A. Bierilo K.K. Flattery A.M. Wong K.K. Castro-Perez J.M. et al.A rapid method for cross-species quantitation of apolipoproteins A1, B48 and B100 in plasma by ultra-performance liquid chromatography/tandem mass spectrometry.Rapid Commun. Mass Spectrom. 2012; 26: 101-108Crossref PubMed Scopus (49) Google Scholar, 9Zhou H. Castro-Perez J. Lassman M.E. Thomas T. Li W. McLaughlin T. Dan X. Jumes P. Wagner J.A. Gutstein D.E. et al.Measurement of apo(a) kinetics in human subjects using a microfluidic device with tandem mass spectrometry.Rapid Commun. Mass Spectrom. 2013; 27: 1294-1302Crossref PubMed Scopus (28) Google Scholar, 10Lassman M.E. McLaughlin T.M. Zhou H. Pan Y. Marcovina S.M. Laterza O. Roddy T.P. Simultaneous quantitation and size characterization of apolipoprotein(a) by ultra-performance liquid chromatography/mass spectrometry.Rapid Commun. Mass Spectrom. 2014; 28: 1101-1106Crossref PubMed Scopus (35) Google Scholar, 11Ceglarek U. Dittrich J. Becker S. Baumann F. Kortz L. Thiery J. Quantification of seven apolipoproteins in human plasma by proteotypic peptides using fast LC-MS/MS.Proteomics Clin. Appl. 2013; 7: 794-801Crossref PubMed Scopus (32) Google Scholar). Although promising, one analytical challenge is to use this method in a single run analysis for the determination of concentrations and tracer enrichments of a significant set of plasma proteins with large differences in molecular mass or abundances (6Pan Y. Zhou H. Mahsut A. Rohm R.J. Berejnaia O. Price O. Chen Y. Castro-Perez J. Lassman M.E. McLaren D. et al.Static and turnover kinetic measurement of protein biomarkers involved in triglyceride metabolism including apoB48 and apoA5 by LC/MS/MS.J. Lipid Res. 2014; 55: 1179-1187Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 11Ceglarek U. Dittrich J. Becker S. Baumann F. Kortz L. Thiery J. Quantification of seven apolipoproteins in human plasma by proteotypic peptides using fast LC-MS/MS.Proteomics Clin. Appl. 2013; 7: 794-801Crossref PubMed Scopus (32) Google Scholar). We recently published an LC/MS/MS method to simultaneously measure the concentration, tracer enrichment, and average size of Apo(a) (9Zhou H. Castro-Perez J. Lassman M.E. Thomas T. Li W. McLaughlin T. Dan X. Jumes P. Wagner J.A. Gutstein D.E. et al.Measurement of apo(a) kinetics in human subjects using a microfluidic device with tandem mass spectrometry.Rapid Commun. Mass Spectrom. 2013; 27: 1294-1302Crossref PubMed Scopus (28) Google Scholar, 12Croyal M. Ouguerram K. Passard M. Ferchaud-Roucher V. Chétiveaux M. Billon-Crossouard S. de Gouville A.C. Lambert G. Krempf M. Nobécourt E. Effects of extended-release nicotinic acid on apolipoprotein (a) kinetics in hypertriglyceridemic patients.Arterioscler. Thromb. Vasc. Biol. 2015; 35: 2042-2047Crossref PubMed Scopus (40) Google Scholar). In the present study, we aimed to describe the development and the validation of a multiplexed LC/MS/MS method, performing in a single run the enrichment measurements and the quantification of six major human Apos (ApoA-I, ApoA-II, ApoB100, ApoC-II, ApoC-III, and ApoE) in plasma samples obtained from a stable isotope kinetic study in humans. UPLC/MS-grade acetonitrile, water, and 99% formic acid were purchased from Biosolve (Valkenswaard, The Netherlands). Ammonium bicarbonate (AB), [5,5,5-2H3]L-leucine, DTT, iodoacetamide, sodium deoxycholate (SDC), trypsin, ammonium hydroxide (NaOH), and 37% hydrochloric acid (HCl) were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France). Synthetic labeled and unlabeled peptides were purchased from Thermo Scientific Biopolymers (Einsteinstrasse, Germany). Six overweight male subjects (age: 46 ± 16 years; body mass index: 31.8 ± 1.5 kg/m2) with hypertriglyceridemia (plasma triglycerides: 208 ± 57 mg/dl) were enrolled. After an overnight fast, each subject received a bolus of 10 μM/kg 2H3-leucine, which allowed a faster plasma enrichment plateau, immediately followed by a constant infusion (10 μM/kg/h) of 2H3-leucine for 14 h. Blood samples were collected at 0, 0.75, 1.5, 2.5, 4, 6, 8, 10, 12, and 14 h in EDTA tubes (Venoject, Paris, France), and the plasma was separated by centrifugation at 4°C for 30 min. Plasma lipoprotein fractions, including VLDL, LDL, IDL, and HDL, were separated by sequential ultracentrifugation methods (13Havel R.J. Eder H.A. Bragdon J.H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6479) Google Scholar, 14Ouguerram K. Chetiveaux M. Zair Y. Costet P. Abifadel M. Varret M. Boileau C. Magot T. Krempf M. Apolipoprotein B100 metabolism in autosomal-dominant hypercholesterolemia related to mutations in PCSK9.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1448-1453Crossref PubMed Scopus (164) Google Scholar) and stored at −80°C until analysis. The Ethics Committee of Nantes University Hospital approved the clinical protocol, and written informed consent was obtained from each subject (reference trial number: NCT01216956). Apo sequences were BLAST searched using the UNIPROT tool (www.uniprot.org), and theoretical proteotypic peptides were searched using the free software peptide mass calculator (http://web.expasy.org/peptide_mass). The peptide candidates were selected to maximize sensitivity, specificity, and stability. Therefore, peptides carrying methionine and cysteine residues were not considered due to potential oxidation and peptides having less than seven amino acids were excluded. Furthermore, peptide candidates had to contain at least one leucine residue for 2H3-leucine enrichment measurement. Each putative candidate was then experimentally sought in the appropriate concentrated lipoprotein fraction and then characterized by LC-high resolution MS (LC/HRMS). The plasma lipoprotein fractions (100 μl) were desalted and concentrated with 50 mM AB buffer (pH 8; 3 ml) and a 5,000 Da molecular mass cut-off filter. The concentrated samples (100 μl) were mixed with 50 mM AB buffer (pH 8; 88 μl), 10% SDC (10 μl), and 500 mM DTT (2 μl). The samples were reduced for 30 min at 60°C, then alkylated with 2 μl of fresh iodoacetamide solution (1 M in 1 M NaOH) for 60 min at room temperature, and protected from light. The samples were digested overnight with 10 μl of trypsin solution (0.1 mg/ml in 1 mM HCl), and 10 μl of 20% formic acid was added to stop the reaction and to precipitate the SDC. Finally, the samples were centrifuged at 15,000 g at 4°C for 15 min, and the supernatants (150 μl) were transferred to vials for LC/MS/MS analyses. Apos were quantified in plasma and plasma lipoprotein fractions, as previously described (12Croyal M. Ouguerram K. Passard M. Ferchaud-Roucher V. Chétiveaux M. Billon-Crossouard S. de Gouville A.C. Lambert G. Krempf M. Nobécourt E. Effects of extended-release nicotinic acid on apolipoprotein (a) kinetics in hypertriglyceridemic patients.Arterioscler. Thromb. Vasc. Biol. 2015; 35: 2042-2047Crossref PubMed Scopus (40) Google Scholar), using synthetic proteotypic peptides for standard solutions and labeled [13C6, 15N2]K or [13C6, 15N4]R synthetic peptides as internal standards (Table 1). Apo quantification was achieved in three replicates and at three kinetic time points (baseline, 6 h, and 14 h).TABLE 1Summary of the analytical parameters selected for the detection of AposApoPeptide SequenceFragmentCone/Collision (V)MRM Transition (m/z)Retention Time (min)ApoA-IM0ATEHLSTLSEKy6+30/25 V608.3 → 664.31.6 ± 0.1M3ATEH L ST L SEK609.8 → 664.3 + 667.3ISATEHLSTLSEK612.3 → 672.3ApoA-IIM0SPELQAEAKy6+30/30 V486.8 → 659.31.4 ± 0.1M3SPE L QAEAK488.3 → 662.3ISSPELQAEAK490.3 → 667.3ApoB100M0NLQNNAEWVYQGAIRy6+50/30 V888.5 → 707.42.7 ± 0.1M3N L QNNAEWVYQGAIR890.0 → 707.4ISNLQNNAEWVYQGAIR893.5 → 717.4ApoC-IIM0TAAQNLYEKy4+80/30 V1,037.9 → 552.31.4 ± 0.1M3TAAQN L YEK1,040.9 → 555.3ISTAAQNLYEK1,045.9 → 560.3ApoC-IIIM0DALSSVQESQVAQQARy8+40/35 V858.9 → 887.52.0 ± 0.1M3DA L SSVQESQVAQQAR860.4 → 887.5ISDALSSVQESQVAQQAR863.4 → 897.5ApoEM0LGPLVEQGRy5+25/30 V484.8 → 588.32.1 ± 0.1M3L GP L VEQGR486.3 → 588.3ISLGPLVEQGR489.3 → 598.3Underlined L indicates the putative incorporation site(s) of 2H3-leucine. Bold indicates the labeled amino acid [13C6, 15N2]K or [13C6, 15N4]R. IS, internal standard. Open table in a new tab Underlined L indicates the putative incorporation site(s) of 2H3-leucine. Bold indicates the labeled amino acid [13C6, 15N2]K or [13C6, 15N4]R. IS, internal standard. Peptide candidates were identified and characterized using a LC/HRMS system composed of a Synapt G2 HDMS® quadrupole-TOF mass spectrometer (Waters Corporation, Milford, MA) with an ESI interface and an Acquity H-Class® UPLCTM device (Waters Corporation). High throughput analyses were then performed on a Xevo® triple-quadrupole mass spectrometer with an ESI interface equipped with an Acquity H-Class® UPLCTM device. Data acquisition and analyses were performed with MassLynx® and TargetLynx® software, respectively (version 4.1; Waters Corporation). Labeled and unlabeled peptides were separated on an Acquity® BEH C18 column (2.1 × 100 mm, 1.7 μm, Waters) at 60°C with a linear gradient of mobile phase B (acetonitrile containing 0.1% formic acid) in mobile phase A (5% acetonitrile in water containing 0.1% formic acid) at a flow rate of 600 μl/min. Mobile phase B was linearly increased from 1 to 50% for 5 min, kept constant for 1 min, returned to the initial condition over 1 min, and kept constant for 1 min before the next injection. Ten microliters of each sample was injected into the LC column. Labeled and unlabeled peptides were then detected by the mass spectrometer with the ESI interface operating in the positive ion mode (capillary voltage, 4 kV; desolvation gas (N2) flow and temperature, 1,000 l/h and 400°C; source temperature, 120°C). The multiple reaction monitoring (MRM) mode was applied for MS/MS detection, and the parameters were optimized for each peptide from synthetic peptide solutions. Selected MRM transitions, cone voltages, and collision energies are described in Table 1. Isolation and measurement of leucine enrichment in ApoB100 and ApoA-I were described previously (14Ouguerram K. Chetiveaux M. Zair Y. Costet P. Abifadel M. Varret M. Boileau C. Magot T. Krempf M. Apolipoprotein B100 metabolism in autosomal-dominant hypercholesterolemia related to mutations in PCSK9.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1448-1453Crossref PubMed Scopus (164) Google Scholar, 15Chétiveaux M. Ouguerram K. Zair Y. Maugère P. Falconi I. Nazih H. Krempf M. New model for kinetic studies of HDL metabolism in humans.Eur. J. Clin. Invest. 2004; 34: 262-267Crossref PubMed Scopus (18) Google Scholar). Briefly, ApoB100- and ApoA-I-containing lipoprotein fractions were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then hydrolyzed with HCl. Amino acids were purified by cation exchange chromatography, derivatized (N-propanol-acetyl chloride and heptafluorobutyric acid), and analyzed by GC/MS to determine [5,5,5-2H3]leucine enrichment. ApoA-I concentrations were measured by immunonephelometry (Behring, Rueil Malmaison, France), and ApoB100 concentrations were obtained by selective precipitation and GC/MS with norleucine as internal standard (16Beghin L. Duhal N. Poulain P. Hauw P. Lacroix B. Lecerf J.M. Bonte J.P. Fruchart J.C. Luc G. Measurement of apolipoprotein B concentration in plasma lipoproteins by combining selective precipitation and mass spectrometry.J. Lipid Res. 2000; 41: 1172-1176Abstract Full Text Full Text PDF PubMed Google Scholar). The M3/M0 ratios were calculated using the chromatographic peak areas, where M3 corresponds to the 2H3-leucine-labeled peptide, and M0 corresponds to the unlabeled peptide. For peptides containing two leucines (ApoA-I and ApoE), two M3 isotopomers could form and be detected simultaneously by the selected MRM transitions. Their proportions were assumed to be identical, as described previously (9Zhou H. Castro-Perez J. Lassman M.E. Thomas T. Li W. McLaughlin T. Dan X. Jumes P. Wagner J.A. Gutstein D.E. et al.Measurement of apo(a) kinetics in human subjects using a microfluidic device with tandem mass spectrometry.Rapid Commun. Mass Spectrom. 2013; 27: 1294-1302Crossref PubMed Scopus (28) Google Scholar, 17Brunengraber H. Kelleher J.K. Des Rosiers C. Applications of mass isotopomer analysis to nutrition research.Annu. Rev. Nutr. 1997; 17: 559-596Crossref PubMed Scopus (94) Google Scholar), and the analytical signal obtained during labeled ApoA-I and ApoE detection was experimentally enhanced 2-fold by the two coeluted isotopomers. In addition, the peptide isotopologues of ApoA-I and ApoE, containing two labeled leucine residues, were not detected in our analytical conditions and were considered as negligible. Therefore, the M3/M0 ratios measured in the biological samples were corrected by dividing the primary result by two, as described previously (17Brunengraber H. Kelleher J.K. Des Rosiers C. Applications of mass isotopomer analysis to nutrition research.Annu. Rev. Nutr. 1997; 17: 559-596Crossref PubMed Scopus (94) Google Scholar). The M3/M0 ratios measured at baseline were subtracted from the following time point ratios. Chromatographic peaks having signal-to-noise ratios below the limit of quantification of 10:1 were excluded and samples were reanalyzed using higher sample volumes before the concentration step (400 μl). Apo concentrations were calculated using calibration curves plotted from standard solutions, as described previously (12Croyal M. Ouguerram K. Passard M. Ferchaud-Roucher V. Chétiveaux M. Billon-Crossouard S. de Gouville A.C. Lambert G. Krempf M. Nobécourt E. Effects of extended-release nicotinic acid on apolipoprotein (a) kinetics in hypertriglyceridemic patients.Arterioscler. Thromb. Vasc. Biol. 2015; 35: 2042-2047Crossref PubMed Scopus (40) Google Scholar). The primary results were expressed in nanomoles and were converted to milligrams per deciliter assuming 1 mol of peptide equal to 1 mol of protein (10Lassman M.E. McLaughlin T.M. Zhou H. Pan Y. Marcovina S.M. Laterza O. Roddy T.P. Simultaneous quantitation and size characterization of apolipoprotein(a) by ultra-performance liquid chromatography/mass spectrometry.Rapid Commun. Mass Spectrom. 2014; 28: 1101-1106Crossref PubMed Scopus (35) Google Scholar). Kinetic analysis was achieved using the SAAM II modeling program (Epsilon Group, Charlottesville, VA). HDL-ApoA-I, HDL-ApoE, and VLDL-ApoE fractional catabolic rates (FCRs) were estimated from the 14 h samples with a mono-compartmental model, as described previously (18Frénais R. Ouguerram K. Maugeais C. Mahot P. Maugère P. Krempf M. Magot T. High density lipoprotein apolipoprotein AI kinetics in NIDDM: a stable isotope study.Diabetologia. 1997; 40: 578-583Crossref PubMed Scopus (79) Google Scholar, 19Bach-Ngohou K. Ouguerram K. Frénais R. Maugère P. Ripolles-Piquer B. Zaïr Y. Krempf M. Bard J.M. Influence of atorvastatin on apolipoprotein E and AI kinetics in patients with type 2 diabetes.J. Pharmacol. Exp. Ther. 2005; 315: 363-369Crossref PubMed Scopus (31) Google Scholar). We applied the same model for HDL-ApoA-II, HDL-ApoC-III, and VLDL-ApoC-III according to Batal et al. (20Batal R. Tremblay M. Barrett P.H. Jacques H. Fredenrich A. Mamer O. Davignon J. Cohn J.S. Plasma kinetics of apoC-III and apoE in normolipidemic and hypertriglyceridemic subjects.J. Lipid Res. 2000; 41: 706-718Abstract Full Text Full Text PDF PubMed Google Scholar) and Chan et al. (21Chan D.C. Watts G.F. Nguyen M.N. Barrett P.H. Factorial study of the effect of n–3 fatty acid supplementation and atorvastatin on the kinetics of HDL apolipoproteins A-I and A-II in men with abdominal obesity.Am. J. Clin. Nutr. 2006; 84: 37-43Crossref PubMed Scopus (75) Google Scholar), but also for HDL-ApoC-II and VLDL-ApoC-II. Kinetic data of VLDL-, IDL-, and LDL-ApoB100 were calculated using a three compartmental model, as previously described (22Maugeais C. Ouguerram K. Krempf M. Maugeais P. Gardette J. Bigot E. Magot T. A minimal model using stable isotopes to study the metabolism of apolipoprotein B-containing lipoproteins in humans.Diabetes Metab. 1996; 22: 57-63PubMed Google Scholar). Plasma leucine was used as precursor pool and pool sizes were considered to be constant, as no significant variation was observed on Apo concentrations at different sampling times (not shown). According to this steady state model, the FCR was equal to the fractional synthetic rate. Production rates (PRs) were calculated by the product of the FCR and the pool sizes of Apos in plasma lipoprotein fractions, assuming a plasma volume of 4.5% of body weight. For ApoE, ApoC-II, and ApoC-III, the concentrations that were not recovered (in the bottom fractions) were considered to be predominantly HDL Apos and these amounts were mathematically added to HDL for calculation of HDL pool sizes (20Batal R. Tremblay M. Barrett P.H. Jacques H. Fredenrich A. Mamer O. Davignon J. Cohn J.S. Plasma kinetics of apoC-III and apoE in normolipidemic and hypertriglyceridemic subjects.J. Lipid Res. 2000; 41: 706-718Abstract Full Text Full Text PDF PubMed Google Scholar). To assess the intra- and inter-assay variability of the LC/MS/MS method, pooled plasma lipoprotein fractions were prepared by mixing 400 μl of plasma lipoprotein fractions from the six subjects at the following kinetic time points: baseline, 45 min, 6 h, and 14 h. These points were chosen to reach baseline, low, intermediate, and high tracer enrichment levels, respectively. The VLDL fractions were used for ApoB100 assay validation, and the HDL fractions were used for the other Apos (i.e., ApoA-I, ApoA-II, ApoC-II, ApoC-III, and ApoE). Each pool was then divided into 18 equal fractions of 100 μl and treated as described above. Six fractions per time point were analyzed, and the analyses were repeated on three consecutive days. The intra- and inter-assay variability of the LC/MS/MS method was calculated [coefficient of variation (CV), percent] with a maximum tolerance level of 15% (23Viswanathan C.T. Bansal S. Booth B. DeStefano A.J. Rose M.J. Sailstad J. Shah V.P. Skelly J.P. Swann P.G. Weiner R. Quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays.Pharm. Res. 2007; 24: 1962-1973Crossref PubMed Scopus (614) Google Scholar). To assess the accuracy of the LC/MS/MS method, enrichment of ApoA-I in the HDL sample and ApoB100 in the VLDL, IDL, and LDL samples were measured by conventional methods using GC/MS (six subjects, 10 kinetic time points per plasma lipoprotein fraction). The paired results, baseline excluded, obtained for ApoA-I (HDL, n = 54) and ApoB100 (VLDL, IDL, and LDL, n = 162) with both analytical methods were analyzed using a Pearson correlation test. A Bland-Altman plot was also generated to test the similarity of both methods accurately (24Bland J.M. Altman D.G. Statistical methods for assessing agreement between two methods of clinical measurement.Lancet. 1986; 1: 307-310Abstract PubMed Scopus (39374) Google Scholar). Graphics and statistical analyses were achieved with GraphPad Prism software (version 6.0; GraphPad Software Inc., La Jolla, CA). The selection of specific peptide markers was performed successfully for each target Apo by LC/HRMS. The in silico selection of the proteotypic peptides led to 2 (ApoA-II and ApoC-III), 3 (ApoC-II), 16 (ApoE), 19 (ApoA-I), and 73 (ApoB100) peptide candidates. The most specific and detectable of them were selected to optimize the assay sensitivity and specificity. As shown in Table 1, each candidate was detected as a doubly charged precursor ion, except for ApoC-II. After MS/MS fragmentations, each precursor ion yielded between 10 (ApoE) and 29 (ApoC-III) specific and singly charged product ions (supplementary Fig. 1A, B). As an example, for ApoA-I and ApoB100, the complete characterization of the fragmentation patterns identified the peptide sequences underlying their specificity. The most intense product ion was then selected for MRM transitions leading to the specific detection of the target peptides (supplementary Fig. 1C). As illustrated for ApoE (supplementary Fig. 2), the MRM mode allowed the specific detection of both labeled and unlabeled target peptides. As expected, the chromatographic peak intensities corresponding to the labeled ApoE peptide (M3) increased during the course of the labeled tracer perfusion. As illustrated in Fig. 1A, the data obtained with the two methods were not statistically different and were significantly correlated with a slope close to one for ApoA-I in HDL (r = 0.994, P < 0.0001, y = 0.99x + 0.01) and ApoB100 in VLDL/IDL/LDL (r = 0.999, P < 0.0001, y = 1.001x + 0.013). For the Bland-Altman plot, the mean difference and the limits of agreement, corresponding to the 95% confidence level (i.e., mean ± 1.96 × SD), were drawn (Fig. 1B), and 94 and 98% of the points were between the limits of similarity for ApoA-I and ApoB100, respectively. The accuracy of the LC/MS/MS method was established by comparing the ApoA-I and ApoB100 enrichment measurements with those obtained by the reference methods used for many years, as described above. The precision of the LC/MS/MS measurements was determined by CVs calculated from six replicates per enrichment level and over three distinct experiments. The intra- and inter-assay variability did not exceed 10.7 and 12.5%, respectively, for any of the Apos or for various enrichments ranging from 0.04 to 7.72% (supplementary Table 1). Finally, the LC/MS/MS efficiency is illustrated in Fig. 2, which shows the kinetic enrichment curves of the six Apos. The kinetic measurements were assessed simultaneously in 240 samples within 1 week (6 subjects, 10 kinetic time points, and 4 plasma lipoprotein fractions). Apos were successfully quantified in plasma and lipoprotein fractions by LC/MS/MS. Total recoveries (mean ± SEM) of ApoE, ApoC-II, and ApoC-III in plasma lipoprotein fractions separated by ultracentrifugation were 69.7 ± 2.5%, 79.1 ± 6.9%, and 67.5 ± 2.7%, respectively, and ranged from 96.2 to 104.7% for ApoA-I, ApoA-II, and ApoB100. As CVs did not exceed 12.4% and Apo concentrations were stable throughout the kinetics, the mean values were kept as final concentrations (Table 2). ApoA-I/ApoA-II concentration ratio was of 4.0 ± 0.7 in HDL and ApoC-III/ApoC-II concentration ratios were of 3.3 ± 0.3 and 3.3 ± 0.9 in HDL and VLDL, respectively. As shown in Table 2, ApoA-I and ApoB100 concentrations measured by LC/MS/MS and conventional methods were not different. Kinetic parameters of Apos were also similar with both methods for ApoB100 and ApoA-I. Compared with previous reported data, the results obtained for ApoA-II, ApoC-II, ApoC-III, and ApoE were in similar ranges (Table 2, supplementary Table 2).TABLE 2Kinetic parameters of AposAposConcentration (mg/dl)FCR (pool/day)PR (mg/kg/day)HDL-ApoA-I, LC/MS/MS139.0 ± 10.50.28 ± 0.0417.7 ± 3.0HDL-ApoA-I, GC/MS136.3 ± 7.60.28 ± 0.0417.2 ± 2.6HDL-ApoA-II, LC/MS/MS38.8 ± 6.40.36 ± 0.066.0 ± 1.0VLDL-ApoB100, LC/MS/MS8.8 ± 1.15.44 ± 1.120.7 ± 2.8VLDL-ApoB100, GC/MS8.7 ± 1.25.65 ± 0.921.3 ± 2.8IDL-ApoB100, LC/MS/MS3.9 ± 0.55.66 ± 1.1510.0 ± 2.5IDL-ApoB100, GC/MS3.7 ± 0.45.60 ± 1.129.9 ± 2.6LDL-ApoB100, LC/MS52.5 ± 8.30.30 ± 0.047.0 ± 1.3LDL-ApoB100, GC/MS49.5 ± 6.50.31 ± 0.056.8 ± 1.3HDL-ApoC-II, LC/MS/MS1.5 ± 0.31.39 ± 0.300.9 ± 0.2VLDL-ApoC-II, LC/MS/MS5.4 ± 1.51.44 ± 0.453.3 ± 1.1HDL-ApoC-III, LC/MS/MS4.5 ± 0.91.11 ± 0.123.2 ± 0.8VLDL-ApoC-III, LC/MS/MS13.2 ± 1.71.17 ± 0.1813.0 ± 2.0HDL-ApoE, LC/MS/MS2.5 ± 0.50.58 ± 0.090.7 ± 0.3VLDL-ApoE, LC/MS/MS1.6 ± 0.32.70 ± 0.462.0 ± 0.6Values are mean ± SEM. 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